HIV remains a global health threat. Replication of the HIV viral genomic RNA occurs by reverse transcription to a double-stranded DNA copy that integrates into the host genome. Reverse transcription is performed by a viral reverse transcriptase, which is both an RNA-dependent and DNA-dependent RNA polymerase. Initiation of reverse transcription occurs at a specific locus at the 5'end of the viral genome, using a complex between viral RNA and a host tRNALys3. Biochemical and in vivo experiments have defined the basic features of the reverse transcription initiation process, and have shown that initiation is the rate-limiting step of reverse transcription and the target of many reverse transcriptase inhibitors of extreme therapeutic importance. However, details of how initiation occurs, is regulated, and inhibited are not known. Here, a combined biophysical and structural approach is proposed to determine the essential molecular features of this central process in HIV replication. In specific aim 1, NMR will be used to define the structure of the viral RNA-tRNALys3 complex, and its mechanism of formation (specific aim 2); these will be challenging RNA NMR problems. The structure of the tRNA-viral RNA-reverse transcriptase ternary complex will be determined by x-ray crystallography (specific aim 3). To complement static structural experiments, the conformational dynamics of RNA that underlie reverse transcription initiation will be delineated using single-molecule fluorescence spectroscopy in specific aim 4. Finally, the structural, thermodynamic, and mechanistic consequences of binding of non-nucleoside RT inhibitors on the initiation complex (specific aim 5). The results of this proposal will have direct impact on our understanding of reverse transcription, and the design of novel strategies to inhibit viral replication.